The global incidence of mosquito-borne viruses such as dengue virus has increased dramatically with the spread of Aedes aegypti mosquitos around the world making it an emerging disease. Approximately 40% of the world's population lives in areas at risk of dengue transmission. Yet there are no approved antivirals for dengue, which can cause a potentially lethal complication referred to as dengue hemorraghic fever or severe dengue. This has become the leading cause of death among children in Asian and Latin American countries endemic for dengue. Our proposed studies will investigate how dengue virus subverts cellular proteins and ribosomes to generate viral proteins. The mechanism of translation initiation is an understudied area of research. Although dengue caps its viral RNA, there are a number of studies that have suggested that dengue uses a cap-independent mechanism of initiation. We will determine how dengue recruits the cellular translational machinery using genetic, biochemical and cell biology approaches. Briefly, we will determine if dengue can recruit 40S subunits directly to the viral RNA and where this recruitment occurs, if it relies on other translation factors or RNA binding proteins. In addition, we will determine whether dengue virus uses both a cap-dependent and a non-canonical mechanism of initiating translation and whether there is a switch during viral infection between these mechanisms. The ribosomal protein S25 (eS25/RPS25) has been shown to be required for diverse non-canonical mechanisms of initiation but not for cap-dependent translation. Therefore, we will use a number of mutants of eS25 to determine whether eS25 is required for dengue translation and what its role is. We have identified the most extensive list of RNA binding proteins that are bound to the dengue viral RNA during a live infection in cells. We will determine if these proteins are required for dengue virus translation and if so where they bind on the dengue virus RNA using a crosslinking-immunoprecipitation method (CLiP-seq). These studies will fundamentally change our understanding of how dengue is translated and the role of cellular RNA binding proteins in this process.